Abstract Complex and lengthy treatment regimens coupled with rising drug resistance underscore the urgent need for new and better drugs to treat tuberculosis (TB) caused by the bacterium Mycobacterium tuberculosis (Mtb). Current treatment regimens for drug resistant TB involve the use of costly, less effective, and toxic drugs that must be continued for up to 24 months. The work proposed here focuses on a proprietary triazolothiadiazole series of narrow spectrum anti-tubercular agent, which demonstrates exclusive selectivity against Mtb with little or no activity against other eubacteria. Due to such high Mtb-selectivity, the triazolothiadiazoles are expected to minimally impact the gut microbiome without promoting selection of cross resistance in non-targeted species during protracted treatment regimens; therefore, this series represent an exciting starting point for the development of an entirely novel class of anti-tubercular agent to specifically treat drug-resistant TB. The unique anti-microbial profile of the triazolothiadiazoles was initially confirmed with three de novo derivatives of the same scaffold, providing robust verification of the Mtb selectivity. The minimal inhibitory concentration (MIC) values of 44 additional de novo derivatives range from 0.05 to 6.25 g/ml against drug-susceptible and multi-drug resistant (MDR) Mtb clinical isolates. Activity against intracellular Mtb replicating within macrophages shows bacteriostatic and bacteriocidal effects at 0.1 g/ml and 10 g/ml. This series has excellent spontaneous resistance frequencies of <10-9 and minimal cytotoxicity in mammalian cell lines with therapeutic index of ?50. However, metabolite profiling in liver microsomes revealed rapid metabolism via N-oxidation and demethylation of an amine moiety in the triazolothiadiazole core. The immediate goal of this Phase I project is to obtain analogs with improved metabolic stability while retaining potent Mtb activity for in vivo efficacy evaluation in the TB mouse model. For the current project, we have developed a medicinal chemistry plan highly focused on resolving metabolic instability. Approximately 50 analogs containing stable substitutions at the metabolically vulnerable spot will be synthesized and profiled in microbiological assays against extracellular and intracellular Mtb along with stability assessment in human and mouse liver microsomes. Our goal is to obtain 3 to 4 potent (MIC <0.5 g/ml) analogs with acceptable microsomal stability in human and mouse liver microsomes (extraction ratio ?30%) for pharmacokinetic experiments and efficacy evaluations in the mouse TB model. A successful outcome for the current Phase I project is a quantitative evidence of treatment-induced suppression of bacterial replication within the lungs of Mtb-infected mice to support the continued advancement of this series in a future Phase II project.